The present invention relates to the in vitro culture of animal cells and, more particularly, to an apparatus for oxygenating culture medium employed in the in vitro culture of animal cells.
The in vitro culture of animal cells, particularly for purposes of recovering proteins either normally secreted by such cells or secreted by such cells by virtue of manipulation of their genetic machinery, has assumed increasingly greater prominence as a consequence of the increasing need for large quantities of proteins for therapeutic, diagnostic and investigative purposes, and the recognition that animal cells (per se, or as a hybrid partner, or as a host for an exogeneous gene) offer the best source of proteins which are the same as or closely similar to those actually employed by animals (e.g., humans) in vivo in carrying out regulatory, immune response, and other like functions.
Despite the recognized advantages of, and needs for, in vitro animal cell culture, the culture of cells outside the animal body is a difficult proposition at best, made even more difficult by the present-day demand that such processes be carried out efficiently and economically so as to achieve ultimate protein products which are not unreasonably expensive. The ultimate aim of in vitro animal cell culture processes is to provide the cells with an environment which closely mimics that which the cells are exposed to in vivo, in terms, e.g., of nutritional requirements, oxygen requirements, temperature, pH, carrying away of wastes, etc., thereby permitting the cells to grow, behave and produce product as they would in vivo, with the added burden of attempting to mimic this environment in larger scale than the micro-environment which normally would be present, for these cells, in the animal itself. At least in theory, it is possible to devise elaborate in vitro systems involving simulations of capillaries, lungs, kidneys and the like to provide the requisite environment, but often not in any remotely cost-effective manner.
A great many in vitro animal cell culture devices and systems are known in the art for culture of both anchorage-dependent cells and cells which can be grown without need for attachment to a substrate. These devices and systems run the gamut from small-scale flasks or roller bottles to somewhat larger scale hollow-fiber reactors, stirred tank reactors, packed bed reactors, and the like. In each, the cells are bathed or submerged in a liquid culture medium which provides the cells with essential nutrients for growth and maintenance and into which the cells secrete products, including protein products of interest.
Among the most important "nutrients" for animal cells is oxygen, and the provision of means for supplying the required degree of oxygen to the culturing cells not only is among the most difficult aspects of cell culturing, but may indeed act to restrict or dictate choice among otherwise potentially available cell culture devices or systems and their scale of feasible operation.
One means for supplying oxygen to cells in culture is by means of surface aeration, i.e., providing oxygen or oxygen-containing gas in the headspace, above the culture medium level, in a closed culture system. Generally, however, the rate at which oxygen can diffusively transfer from the gas phase to the liquid phase in such systems is relatively low and, thus, growth and maintenance of only a relatively small number of cells can be supported in this manner, relegating it to utility only in small flasks or vessels. The rate of gas transfer can be increased if the liquid phase is agitated (e.g., as in a stirred reactor), but here again the increase is not so great as to offer utility in anything other than relatively small systems.
Another means for providing oxygen to cells in culture medium is to bubble gas directly through the culture (sparging). While this is a very efficient means of oxygenation, it generally is very damaging to animal cells. Also, sparging leads to foam formation which itself can damage the cells. Although the use of surfactants can eliminate or suppress foam formation, the presence of the surfactant in the eventually harvested culture medium can lead to very difficult and expensive problems in purification of the desired secreted protein product.
It also has been proposed to provide oxygen to cells submerged in culture medium (e.g., as in a stirred-tank reactor) by indirect sparging, i.e., passing oxygen into or on one side of a gas-permeable (but generally liquid-impermeable) tube or membrane arranged in the medium (e.g., silicone rubber tubes or sheets), and through or across which the oxygen permeates into the culture medium. It is generally possible in this way to achieve bubble-free and foam-free aeration, although gassing efficiency is not as high as in direct sparging.
Another means for providing oxygen to cells is to continuously or intermittently remove culture medium from the system, separately gasify the medium to provide it with oxygen, and then return the medium to the cell culture system where it can give up its oxygen to the cells. Although somewhat inherently limited by the relatively low solubility of oxygen in the culture medium, operation in this manner can prove quite effective, and is often employed in association with cell culture devices (e.g., packed-bed reactors, hollow fiber reactors, fluidized bed reactors and the like) which are designed to retain cells therein while culture medium is removed for oxygenation and recirculated to the reactor. While closed loop systems of this type are operable at free cell suspension densities (i.e., the density of cells not retained in the reactor when medium is withdrawn therefrom) which are significantly lower than is the case with stirred reactors, the removed medium nevertheless generally contains a number of cells which is still sufficiently large to bring about concern for damage to these cells during oxygenation of the medium.
Along these lines, it is known to employ blood oxygenators or other artificial lung-type devices to provide oxygen to culture medium removed (along with a fair number of cells) from a culture device, wherein the oxygen is provided to the medium across gas-permeable, liquid-impermeable membrane surfaces (e.g., in the form of tubes, hollow fibers or sheets), but the devices known heretofore for such purpose prove quite inefficient and/or troublesome, particularly when large-scale, cost-effective culture is at issue. For example, in order to provide oxygenation of the culture medium sufficient to support large-scale culture of cells it is desirable that the rate of mass transfer of gas across the membrane and into the culture medium be as large as possible, and that the available surface area over which mass transfer takes place also be large. At the same time, it is necessary that the oxygenation apparatus be capable of withstanding the rigors of continuous, long-term operation, requiring that the materials be capable of repeated autoclaving or like sterilization cycles and that the apparatus not too easily or readily clog with cells and cell debris contained in the culture medium. These desires and concerns are often self-contradictory, leading to compromise constructions which, e.g., provide large surface areas and thin membranes to optimize mass transfer, but which break down (leading to bubble and foam formation) after more than a single sterilizing cycle; or provide hearty membrane surfaces for strength but have poor mass transfer characteristics and need to be made very large; or provide reasonably-sized yet tightly packed or wound membrane surfaces which readily foul and/or limit medium throughput and/or cause the medium to seek out preferential flow pats therethrough and thus reduce the effective area of gas transfer. As a consequence, none of the constructions and configurations permit of the long-term, cost-effective, efficient oxygenation demanded for commercial-scale in vitro cell culture.